Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses

Definitions

• the disclosure is directed to methods of using dantrolene or a dantrolene prodrug, or a pharmaceutically acceptable salt thereof, to treat coronavirus infections BACKGROUND
• Coronaviruses ( Coromviridae , Orthocoronavirinae) cause diseases in mammals and birds. In humans, the vimses cause respiratory infections, including the common cold. Coronaviruses cause, for example, Severe Acute Respiratory’ Syndrome (SARS), COVID-19, and Middle East Respiratory Syndrome. Coronaviruses pose a threat to worldwide public health. Treatments effective to treat these vimses are needed.
• SARS Severe Acute Respiratory’ Syndrome
• COVID-19 COVID-19
• Middle East Respiratory Syndrome Middle East Respiratory Syndrome
• the disclosure is directed to methods of treating a coronavirus infection in a subject comprising administering to the subject dantrolene or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.
• the disclosure is also directed to methods for inhibiting replication of a coronavirus in a subject comprising administering to the subject dantrolene, or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.
• the disclosure is also directed to methods for inhibiting replication of a coronavirus in a host cell, inhibiting entry of a coronavirus into a host cell, inhibiting a coronavirus virion maturation in a host cell, or inhibiting release of a coronavirus from a host cell compnsing administering to the host cell dantrolene, or a pharmaceutically acceptable
• the disclosure is also directed to methods for reducing the infectivity of a coronavirus by administering to a host cell dantrolene, or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.
• an exemplary embodiment includes from the one particular value and/or to the other particular value. All ranges are inclusive and combinable. Further, reference to values stated in ranges includes each and ever value within that range. When values are expressed as approximations, by use of the preposition “about,” it will be understood that the particular value forms another embodiment.
• the term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass reasonable variations of the value, such as, for example, ⁇ 10% from the specified value. For example, the phrase “about 50%” can include ⁇ 10% of 50, or from 45% to 55%, inclusive of 50%.
• pharmaceutically acceptable indicates that the designated entity such as, for example, a pharmaceutically acceptable excipient, is generally chemically and/or physically compatible with other ingredients in a composition, and/or is generally physiologically compatible with the recipient thereof
• subject(s), “mdividual(s),” and “patient(s)”, refer to mammals, including humans.
• human(s) refers to and includes, a human child, adolescent, or adult.
• treats refer to and include ameliorative, palliative, and/or curative uses and results, or any combination thereof.
• the methods described herein can be used prophylactically. It should be understood that “prophylaxis” or a prophylactic use or result do not refer to nor require absolute or total prevention (i.e., a 100% preventative or protective use or result).
• prophylaxis or a prophylactic use or result refers to uses and results in which administration of a compound or composition diminishes or reduces the severity of a particular condition, symptom, disorder, or disease described herein; diminishes or reduces the likelihood of experiencing a particular condition, symptom, disorder, or disease described herein; or delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein; or any combination of the foregoing.
• terapéutica refers to an amount of a compound or composition that (a) treats a particular condition, symptom, disorder, or disease described herein; (b) attenuates, ameliorates, or eliminates one or more symptoms of a particular condition, disorder, or disease described herein; (c) delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein. It should be understood that the terms “therapeutic” and “therapeutically effective” encompass
• Coronaviruses include, for example, alphacoronavirus (e.g., colacovirus, decacovirus, duvinacovirus, luchacovirus, minacovirus, minunacovirus, myotacovirus, nyctacovirus, pedacovirus, rhinacovirus, setracoviurs, tegocovirus), betacoronavirus (e.g., embecovirus, hibecovirus, merbecoviurs, nobecovirus, sarbecovirus), deltacoronavirus (e.g, andecovirus, bul decovirus, herdecovirus, moordecovirus), and gammacoronavirus (e.g, cegacovirus, igacovirus).
• alphacoronavirus e.g., colacovirus, decacovirus, duvinacovirus, luchacovirus, minacovirus, minunacovirus, myotacovirus, nyctacovirus
• coronaviruses include SARS-CoV-1, SARS-CoV-2, MERS- CoV, PREDICT-CoV-35, PREDICT-CoV-47, PREDICT-CoV-82, PREDICT-CoV-90, PREDICT-CoV-92, PREDICT-CoV-93, and PREDICT-CoV-96.
• a “host cell” is a cell in which a coronavirus can replicate.
• normalization of fever is reduction of a subject’s temperature to ⁇ 36.6 °C armpit, ⁇ 37.2 °C oral, or ⁇ 37.8 °C rectal, sustained for at least 24 hours.
• normalization of oxygen saturation is an increase in a subject’s peripheral capillary oxygen saturation (Sp02) > 94%, sustained for at least 24 hours.
• inhibiting replication of coronavirus refers to decreasing viral load of a coronavirus. Methods for determining coronavirus replication inhibition can be determined by those skilled in the art.
• Dantrolene is approved for treating malignant hyperthermia and preventing malignant hyperthermia in high-risk patients.
• Malignant hyperthermia is a condition that predisposes susceptible individuals to a life-threatening adverse reaction upon exposure to potent volatile anesthetics (halothane, isoflurane, sevoflurane, desflurane, etc.) and the skeletal muscle relaxant succinyl choline.
• the anesthetic drugs trigger an uncontrolled Ca 2+ release from the endoplasmic reticulum (ER) through the ryanodine receptors (RyR) causing a rapid and sustained rise in myoplasmic Ca 2+ .
• Administration of dantrolene reestablishes cellular calcium homeostasis by inhibiting the release channels in the ER, resulting in lower levels of intracellular Ca 2+ .
• RYANODEX (dantrolene sodium, 250 mg/vial) is approved for treating malignant hyperthermia and for preventing malignant hyperthermia in high-risk patients.
• RYANODEX forms an aqueous nanosuspension for IY injection containing dantrolene 50
• 5UB5TITUTE SHEET (RULE 26) mg/mL upon reconstitution with 5 mL of USP sterile water for injection (WFI) (without a bacteriostatic agent). Dissolution of RYANODEX suspension in human plasma is extremely rapid, achieving complete dissolution within 1 minute.
• Dantrolene is a surprisingly effective treatment for a coronavirus infection. Dantrolene can decrease the vims’ ability to replicate, mature, create virions, release from cells, and/or infect other cells.
• Methods of the disclosure can also be accomplished using dantrolene prodrugs, and pharmaceutically acceptable salts thereof.
• dantrolene prodrugs are described in WO2019/079721, the entirety of which is incorporated by reference herein.
• Preferred dantrolene prodrugs include, for example, compounds of formula I wherein R is -P(0)(0H)2 or -P(0)(0RI)(0R2); Ri is H, -Ci-26alkyl, aryl, Ci-6alkC(0)0-Ci- 26alkyl, -Cialk0C(0)Ci-26alkyl, or Cialk0C(0)0Ci-26alkyl; and R2 is -Ci-26alkyl, aryl, Ci- 6alkC(0)0-Ci-26alkyl, -Cialk0C(0)Ci-26alkyl, or Cialk0C(0)0Ci- 26 alkyl, as well as pharmaceutically acceptable salts thereof.
• Particularly preferred compounds of formula I include compounds 2 and 2a:
• dantrolene prodrugs include compounds of formula II wherein R 3 is H, -C(0)-Z-N(R 4 )(R 5 ), -C(0)Z-C(0)-0H, or -C(0)-NH-Y-CH2-0C(0)-Z- C(0)-0H; Z is Ci-6alk; Y is arylene; Ci-6alkyl; Rs is H or Ci-6alkyl; or R 4 and Rs, together with the nitrogen to which they are attached, form a heterocycloalkyl; as well as pharmaceutically acceptable salts thereof.
• One aspect of the invention is directed to methods of treating a coronavirus infection in a subject.
• the subject may be clinically diagnosed with a coronavirus infection.
• Criteria for diagnosing a subject with a coronavirus infection are known and include laboratory confirmation of a coronavirus infection as determined using PCR, in combination with presentation of one or more coronavirus infection symptoms. Other assays for determining a coronavirus infection can also be used.
• the subject may be suspected of having a coronavirus infection, based on, for example, having experienced close contact with another person who has been clinically diagnosed with a coronavirus infection.
• Other subjects may be suspected of having a coronavirus infection based on the subject’s symptom presentation.
• the subject is treated for a coronavirus infection by administering to the subject dantrolene.
• the subject is treated for a coronavirus infection by administering to the subject a pharmaceutically acceptable salt of dantrolene, for example, dantrolene sodium.
• the subject is treated for a coronavirus infection by administering to the subject a dantrolene prodrug, for example, Compound 2.
• the subject is treated for a coronavirus infection by administering to the subject a salt of a dantrolene prodrug, for example, Compound 2a.
• Administration is preferably of a therapeutically effective amount of the dantrolene, pharmaceutically acceptable salt of dantrolene, dantrolene prodrug, or salt of a dantrolene prodrug.
• Therapeutically effective amounts include, for example, about 1 mg/kg to 10 mg/kg, administering daily, for one or more days. Particularly preferred amounts include about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg, administered daily, in one or more doses, for one or more days.
• the administration results in at least a 1 -point decrease in the subject’s WHO Ordinal Scale score, as compared to the subject’s WHO Ordinal Scale score at baseline. Methods of assessing WHO Ordinal Scale score are known in the art. In some aspects, the administration results in a 2-point decrease in the subject’s WHO Ordinal Scale score, as compared to the subject’s WHO Ordinal Scale score at baseline. In some aspects, the administration results in a 3-point decrease in the subject’s WHO Ordinal Scale score, as compared to the subject’s WHO Ordinal Scale score at baseline. In some aspects, the administration results in a 4-point decrease in the subject’s WHO Ordinal Scale score, as compared to the subject’s WHO Ordinal Scale score at baseline. In some aspects, the 6
• 5UB5TITUTE SHEET (RULE 26) administration results in a 5-point decrease in the subject’s WHO Ordinal Scale score, as compared to the subject’s WHO Ordinal Scale score at baseline.
• the administration results in an improvement, for example, an increase in the subject’s Sequential Organ Failure Assessment daily score, as compared to baseline.
• Methods of assessing a subject’s Sequential Organ Failure Assessment daily score are known in the art.
• the administration results in a reduction of time to normalization of fever in the subject, as compared to the amount of time to normalization of fever in a control subject, for example, as compared to a subject who has only received standard of care treatment.
• the administration results in a reduction of fever in the subject treated for a coronavirus infection.
• the administration results in a clinically significant reduction of fever in the subject.
• the administration results in a normalization of fever in the subject.
• the administration results in a reduction of time to normalization of oxygen saturation in the subject, as compared to the amount of time to normalization of oxygen saturation in a control subject, for example, as compared to a subject who has only received standard of care treatment.
• the administration results in an increase of oxygen saturation in the subject.
• the administration results in a clinically significant increase in oxygen saturation in the subject.
• the administration results in normalization of oxygen saturation in the subject
• the administration results in improvement in one or more symptoms of a coronavirus infection in the subject. In other aspects, the administration results in a clinically significant improvement in one or more symptoms of a coronavirus infection in the subject.
• Other aspects of the disclosure are directed to methods of inhibiting replication of a coronavirus in a subject.
• the subject may be clinically diagnosed with a coronavirus infection. Criteria for diagnosing a subject with a coronavirus infection are known and include laboratory confirmation as determined using PCR. Other assays for determining coronavirus infections can also be used.
• the subject may be suspected of having a coronavirus infection, based on, for example, having experienced close contact with another person who has been clinically diagnosed with a coronavirus infection.
• inhibition of coronavirus replication in a subject is accomplished by administering to the subject dantrolene. In other aspects, inhibition of coronavirus replication in a subject is accomplished by administering to the subject a pharmaceutically acceptable salt of dantrolene, for example, dantrolene sodium. In some aspects, inhibition of coronavirus replication in a subject is accomplished by administering to the subject a dantrolene prodrug, for example, Compound 2. In some aspects, inhibition of coronavirus replication in a subject is accomplished by administering to the subject a salt of a dantrolene prodrug, for example, Compound 2a.
• Administration is preferably of a therapeutically effective amount of the dantrolene, pharmaceutically acceptable salt of dantrolene, dantrolene prodrug, or salt of a dantrolene prodrug.
• Therapeutically effective amounts include, for example, about 1 mg/kg to 10 mg/kg, administenng daily, for one or more days. Particularly preferred amounts include about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg, administered daily, in one or more doses, for one or more days.
• Some aspects of the disclosure are directed to methods for inhibiting replication of coronavirus in a host cell. Inhibition of viral replication can be determined by those skilled in the art. In these methods, replication is inhibited by administering dantrolene to the host cell. In other aspects, replication is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, replication is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, replication is inhibited by administering to the host cell a salt of a dantrolene prodrag, for example, Compound 2a.
• Some aspects of the disclosure are directed to methods for inhibiting entry of coronavirus into a host cell. Inhibition of viral entry into a host cell can be determined by those skilled in the art. In these methods, viral entry is inhibited by administering dantrolene to the host cell. In other aspects, viral entry is inhibited by administenng a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, viral entry is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, viral entry is inhibited by administering to the host cell a salt of a dantrolene prodrag, for example, Compound 2a.
• Some aspects of the disclosure are directed to methods for inhibiting coronavirus virion maturation in a host cell. Inhibition of virion maturation in a host cell can 8
• virion maturation is inhibited by administering dantrolene to the host cell.
• virion maturation is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium.
• virion maturation is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2.
• virion maturation is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.
• Some aspects of the disclosure are directed to methods for release of coronavirus from a host cell. Inhibition of release from a host cell can be determined by those skilled in the art. In these methods, viral release is inhibited by administering dantrolene to the host cell. In other aspects, viral release is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, viral release is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, viral release is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.
• Some methods of the disclosure are directed to methods for reducing the infectivity of a coronavirus. Reduction of infectivity can be determined by those skilled in the art. In these methods, infectivity is reduced by administering dantrolene to the host cell.
• infectivity is reduced by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium.
• infectivity is reduced by administering to the host cell a dantrolene prodrug, for example, Compound 2.
• infectivity is reduced by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.
• the study is a single-center, open-label, two-arm parallel study of dantrolene for the adjuvant treatment of COVID-19 administered intravenously (IV).
• IV intravenously
• dantrolene will be administered in conjunction with current standard of care following medical practice and procedures established for the in-hospital treatment of patients with COVID-19.
• subjects will receive current standard of care following medical practice and procedures established for the in-hospital treatment of patients with COVID-19.
• Group B standard of care only Treatment Administration - Group A
• ⁇ A 1 mg/kg dose will be administered as IV push approximately every 12 hours for 2 consecutive days (Study Day 1 and Day 2).
• Each subject participating in the study will receive up to 4 (four) 1 mg/kg doses (Days 1-2) and up to 24 (twenty four) doses of 2 mg/kg (Days 3-14), each administered as an IV push (up to a minute).
• the study will include 2 phases: Screening and Treatment.
• eligible patients will be randomized to Group A or Group B and the Treatment Phase will be initiated and will proceed with administration of Study Drug as indicated above to Group A
• Non- eligible subjects will receive medical assistance as deemed necessary by the attending physician following accepted medical practices.
• Dantrolene toxicity may include muscular weakness and alterations in the state of consciousness (e g., lethargy, sedation), vomiting, diarrhea, and crystalluria, which are not attributable to other cause, such as progression of COYID-19, other underlying conditions (e g., sepsis, hypoxia, uncontrolled diabetes) and/or concomitant medications (e.g., sedatives, antibiotics, antipyretics).
• the study drug can be stopped at any time.
• 5UB5TITUTE SHEET •Has laboratory-confirmed SARS-CoV-2 infection as determined by PCR. or other commercial or public health assay within 48 hours prior to Screening.
• Ryanodex (dantrolene sodium) for injectable suspension; 250 mg/vial to be reconstituted with 5 mL of sterile water for injection (without a bacteriostatic agent) to yield a 50 mg/mL suspension; to be administered as a rapid IV push of 1 mg/kg or 2 mg/kg, as described in the protocol.
• Dantrolene concentrations were tested: 5. 10, 20, 30, 40, 50 and 100 mM (as RYANODEX, dantrolene sodium).
• VN virus neutralization
• VN Virus Neutralization Assay.
• the VN assay was performed using Vero E6 cells which are susceptible to SARS-CoV-2 infection. The cells were seeded into 96-well plates one (1) to three (3) days prior to VN assay, and were incubated at 37°C and 5% CO2. On the day of assay, the monolayer of Vero E6 cells was at least 70% to 80% confluent in order to run the VN. The preparation of the compound for VN was performed the day of assay. The compound was diluted with serum-free media to the desired starting concentration (1:10) and then serially diluted 2-fold in serum-free media.
• Standardization of the virus required that a TCID50 had previously been run to determine the concentration of infectious virus particle per mL of virus stock Using the TCID50 titer, calculations were performed to define how much serum-free media to add to the virus stock to yield le2 TCID50/mL.
• an equal volume of it was added to the deep 96-well plate containing the diluted compound samples. Incubation was maintained for at least one hour. The virus/compound mixture was then transferred from the deep 96-well racks into the appropriate Vero-seeded 96-well plates. After this addition, the plates were returned to the 37°C and 5% CO2 incubator for three (3) to five (5) days to six (6) days.
• the titer was the inverse of the last dilution of dantrolene that inhibits the viral infection (cells that do not display CPE), i.e.. the lowest effective titer was the last dilution of dantrolene that consistently inhibited viral infection (cells do not display CPE) across all time points.
• dantrolene modulates intracellular Ca 2+ , including by mechanisms not previously reported, thereby affecting the ability of the SARS-CoV-2 virus to, for example, infect cells, replicate, mature, create virions, or release from cells.
• Dantrolene concentrations were tested: 5, 10, 20, 30, 40, 50 and 100 mM (as RYANODEX, dantrolene sodium).
• VN virus neutralization
• VN Vims Neutralization Assay.
• the VN assay was performed using Vero E6 cells, an African green monkey cell line, which are susceptible to SARS-CoV-2 infection.
• Vero E6 cells were cultured in growth media (Dulbecco’s Modified Eagle Medium supplemented with 5% FBS (fetal bovine serum), Glutamax, and PSN (penicillin, streptomycin, and neomycin)).
• the cells were seeded into deep 96-well plates one day prior to the VN assay and incubated at 37°C and 5% CO2 to allow the cells to grow to 70% confluency. Each of the samples and controls were performed in triplicate.
• RYANODEX for VN was prepared the day of assay.
• RYANODEX was reconstituted with 5 mL of sterile water for injection, as described in the RYANODEX Prescribing Information, to prepare an initial stock having a dantrolene concentration of 50 mg/mL.
• the stock was further diluted to a dantrolene concentration of 100 mM using cell growth media and then serially diluted to 50, 40, 30, 20, 10 and 5 mM.
• the dilutions were preincubated on cells for 60 minutes prior to addition of vims.
• TCID50 Medium Tissue Culture Infectious Dose
• 5UB5TITUTE SHEET (RULE 26) performed to define how much serum-free media to add to the virus stock to yield le2 TCID50/mL. Once the standardized virus was made, an equal volume of it was added to the deep 96-well plate containing the diluted compound samples. Virus was added to the appropriate wells and incubated with cells and compound for 2 hours. The cells were then washed 3 times with fresh media and 100 mE/well of fresh media was added to all wells and further incubated for 6 days. During the incubation period, the wells were observed under a phase contrast inverted scope and were scored for the presence or absence of SARS-CoV-2 cytopathic effects (CPE) in the cells.
• CPE SARS-CoV-2 cytopathic effects
• the titer was the inverse of the last dilution of dantrolene that inhibits the viral infection (cells that do not display CPE) , i.e., the lowest effective titer was the last dilution of dantrolene that consistently inhibited viral infection (cells do not display CPE) across all time points.
• the VN assay demonstrated the in vitro antiviral activity and lack of cytotoxicity of RYANODEX at dantrolene concentrations compatible with human plasma levels observed after administration of the recommended doses of RYANODEX.
• dantrolene modulates intracellular Ca 2+ , including by mechanisms not previously reported, thereby affecting the ability of the SARS-CoV-2 virus to, for example, infect cells, replicate, mature, create virions, or release from cells.
• the study is a single-center, open-label, two-arm study of dantrolene for the adjuvant treatment of coronavirus infection administered intravenously (IV).
• IV intravenously
• dantrolene will be administered in conjunction with current standard of care
• 5UB5TITUTE SHEET (RULE 26) following medical practice and procedures established for the treatment of patients with a coronavirus infection.
• subjects will receive current standard of care following medical practice and procedures established for the treatment of patients with a coronavirus infection.
• Group B standard of care only Treatment Administration - Group A
• Eligible coronavirus infection subjects randomized to Group A will receive dantrolene (as RYANODEX, dantrolene sodium) as follows:
• the study will include 2 phases: Screening and Treatment.
• eligible patients will be randomized to Group A or Group B and the Treatment Phase will be initiated and will proceed with administration of Study Drug as indicated above to Group A
• Non- eligible subjects will receive medical assistance as deemed necessary by the attending physician following accepted medical practices.
• Dantrolene toxicity may include muscular weakness and alterations in the state of consciousness (e.g., lethargy, sedation), vomiting, diarrhea, and crystalluria, which are not attributable to other cause, such as progression of coronavirus infection, other underlying conditions (e.g., sepsis, hypoxia, uncontrolled diabetes) and/or concomitant medications (e.g., sedatives, antibiotics, antipyretics).
• the study drug can be stopped at any time.
• ALT Alanine Aminotransferase
• AST aspartate aminotransferase
• UPN upper limit of normal
• Ryanodex (dantrolene sodium) for injectable suspension; 250 mg/vial to be reconstituted with 5 mL of sterile water for injection (without a bacteriostatic agent) to yield a 50 mg/mL suspension; to be administered as a rapid IV push of 1 mg/kg or 2 mg/kg, as described in the protocol.
• Dantrolene concentrations are tested: 5, 10, 20, 30, 40, 50 and 100 mM (as RYANODEX, dantrolene sodium).
• VN virus neutralization
• VN Virus Neutralization Assay.
• the VN assay is performed using cells which are susceptible to coronavirus infection. The cells are seeded into 96-well plates one (1) to three (3) days prior to VN assay, and are incubated at 37°C and 5% CO2. On the day of assay, the monolayer of cells is at least 70% to 80% confluent in order to run the VN. The preparation of the compound for VN is performed the day of assay. The compound is diluted
• Standardization of the virus requires that a TCID50 has previously been run to determine the concentration of infectious virus particle per mL of virus stock Using the TCID50 titer, calculations are performed to define how much serum-free media to add to the virus stock to yield le2 TCID50/mL.
• an equal volume of it is added to the deep 96-well plate containing the diluted compound samples. Incubation is maintained for at least one hour. The virus/compound mixture is then transferred from the deep 96-well racks into the appropriate cell-seeded 96-well plates. After this addition, the plates are returned to the 37°C and 5% CO2 incubator for three (3) to five (5) days to six (6) days.
• CPE coronavirus cytopathic effects
• dantrolene modulates intracellular Ca 2+ , including by mechanisms not previously reported, thereby affecting the ability of coronaviruses to, for example, infect cells, replicate, mature, create virions, or release from cells.
• VN virus neutralization
• VN assays require a compatible host cell that is susceptible to virus infection.
• Suitable cells lines include, but are not limited to, Vero E6, HeLa, 293T, L929, fibroblasts, CHO, B95-8, MRC-5, HEp-2, SPF CE, MDCK, COS-7, and LLC-MK2 Derivative.
• primary cells isolated from an animal can be used in the assay.
• Cells used for VN assays can be wild-type cells, or they can be cells that have been genetically modified using techniques known in the art.
• Cells lines are cultured in an appropriate growth media, including, but not limited to Dulbecco’s Modified Eagle Medium (DMEM), RPMI, Eagle’s Minimum Essential Medium (EMEM), Leibovitz’s L-15 Medium, and VeroPlus SFM.
• DMEM Modified Eagle Medium
• RPMI Eagle’s Minimum Essential Medium
• EMEM Minimum Essential Medium
• Leibovitz L-15 Medium
• VeroPlus SFM fetal bovine serum
• glutamax penicillin, streptomycin, and neomycin.
• supplements can be added to select for cells with specific genetic modifications.
• the cells are seeded into deep 96-well plates prior to the VN assay and incubated at 37°C and 5% CCh to allow the cells to grow' to an appropriate confluency. Each of the samples and controls are performed in triplicate.
• RYANODEX for VN is prepared the day of assay.
• RYANODEX is reconstituted with 5 mL of sterile water for injection, as described in the RYANODEX Prescribing Information, to prepare an initial stock having a dantrolene concentration of 50 mg/mL.
• the stock is further diluted to a dantrolene concentration of 100 mM using cell growth media and then serially diluted to 50, 40, 30, 20, 10 and 5 mM.
• the dilutions are preincubated on cells for 60 minutes prior to addition of vims.
• TCID50 Medium Tissue Culture Infectious Dose
• 5UB5TITUTE SHEET (RULE 26) Procedures, Serological detection of avian influenza A (H7N9) infections by microneutralization assay, May 23, 2013. Using the TCID50 titer, calculations are performed to define how much serum-free media to add to the virus stock to yield le2 TCID50/mL.
• the standardized virus is made, an equal volume of it is added to the deep 96-well plate containing the diluted compound samples.
• Virus is added to the appropriate wells and incubated with cells and compound for an appropriate amount of time consistent with observations from the TCID50 assay. In some cases, this time period is 2 hours. In other cases, the time period is 30 minutes to 1 hour. In other instances, the time period is 4-8 hours. In yet other instances, the virus may be incubated with the cells for 24-48 hours. And in other instances, cells may continue to be incubated with virus over the duration of the assay. Further, infection may be facilitated by other methods known in the art, such as spin infection.
• the cells are then washed 3 times with fresh media and 100 pL/well of fresh media is added to all wells and further incubated for an appropriate length of time depending on the VN assay being employed.
• the cells are assayed for indications of viral infection and replication using methods known in the art.
• the viral load of cells treated under different conditions may be measured using polymerase chain reaction (PCR) or other methods known in the art.
• PCR polymerase chain reaction
• changes to cellular morphology, such as plaque formation may be assayed.
• wells containing vims are scored for the presence or absence of viral cytopathic effects (VPE) in the cells using methods known in the art, such as light microscopy or Annexin V, FITC, propidium iodide (PI), or haemotoxyhn and eosin (H&E) staining.
• VPE viral cytopathic effects
• chicken red blood cell suspensions are incubated with a serially diluted virus and monitoring for formation of a red blood cell lattice in a hemagglutination assay.
• the attachment of a suspension of red blood cells to the surface of cell monolayers infected with vims is monitored in a hemadsorption assay.
• the titer is the inverse of the last dilution of dantrolene that inhibits the viral infection, i.e., the lowest effective titer is the last dilution of dantrolene that consistently inhibits viral infection across all time points.
• 5UB5TITUTE SHEET (RULE 26)
• dantrolene control that includes RYANODEX alone at the test concentrations without virus to ensure that dantrolene itself at the tested concentrations does not cause a positive signal using the assay for viral infection.
• negative control wells (without RYANODEX or virus) to verify that the serum-free media does not cause a positive signal using the assay for viral infection.
• a back-titer of the virus that includes 100 TCID50/well of virus is performed which acts as a positive VPE or viral infection control, and it serves to verify that the titer of the standardized virus is within acceptable range. Results from the samples on that plate are considered valid if all of these controls meet their acceptance criteria.
• dantrolene modulates intracellular Ca 2+ , including by mechanisms not previously reported, thereby affecting the ability of coronaviruses to, for example, infect cells, replicate, mature, create virions, or release from cells.

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